AKR1C3 SNPs are more common in psoriasis patients than in healthy populations, and are more highly associated with psoriasis vulgaris than with psoriatic arthritis
The patient cohort comprised 231 Japanese psoriasis patients (171 males, 60 females) treated with biologics at Nagoya City University Hospital. The diagnosis of psoriasis was based on clinical manifestations and histopathology. Disease severity was assessed before treatment on the basis of the Psoriasis Area and Severity Index (PASI) score. In this cohort, the mean PASI score was 24.6. Of the 231 patients, 130 had psoriasis vulgaris (PV), 67 had psoriatic arthritis (PsA), 22 had generalized pustular psoriasis (GPP), 5 had erythrodermic psoriasis (EP), and 7 had coexisting PsA and GPP. The body mass index (BMI) of this cohort ranged from 15.6 to 44.1 (mean 25.6). The clinical characteristics of the cohort are summarized in Table 1.
Table 1
Patient characteristics and psoriasis types.
Characteristics
|
|
Cases
|
|
231
|
Race
|
|
Japanese
|
Age at onset (range)
|
|
33.3 (0–79)
|
sex
|
Male
|
171
|
|
Female
|
60
|
BMI at first visit (range)
|
|
25.6 (15.6–44.1)
|
PASI score before treatment (range)
|
24.6 (0.6–72)
|
Tobacco smoking
|
Ever smoker
|
99
|
Never smoker
|
130
|
unknown
|
2
|
Psoriasis type
|
PV
|
130
|
PsA
|
67
|
GPP
|
22
|
EP
|
5
|
PsA + GPP
|
7
|
BMI, body mass index; PV, psoriasis vulgaris; PsA, psoriatic arthritis; GPP, generalized pustular psoriasis; EP, erythrodermic psoriasis; PASI, Psoriasis Area and Severity Index
A TaqMan genotyping assay was performed using DNA isolated from whole blood. Rs12529 and rs12387, 2 common AKR1C3 SNPs, were examined. The frequency of both SNPs is as low as 6–11% in healthy Japanese. The rs12529 SNP frequency in Japanese differs significantly from the global trend, which has a rs12529 G > C frequency of 42%. The TaqMan SNP genotyping assay revealed that the rs12529 genotype distribution in our cohort was G/G:174 (75.3%), G/C:52(22.5%), and C/C:5 (2.2%). Thus, the probability of a G > C mutation in rs12529 was 13.4%. Surprisingly, the A > G mutation in rs12387 and G > C mutation in rs12529 were always observed simultaneously. The proportions of rs12529 G > C SNPs and rs12387A > G SNPs were significantly higher in the patient cohort than in the 2 cohorts of healthy Japanese individuals in the NCBI database (rs12529, p = 0.0080 [vs ss69068306], p = 0.0029 [vs ss71643788], chi-square test, Table 2, and rs12387, p = 0.0093 [vs ss2827707], p = 0.0026 [vs ss66361131], chi-square test, Table 3). Compared with a larger cohort of Japanese called "8.3K JPN”, which may also include a variety of diseases including psoriasis, the rs12529 G > C SNP tended to be more prevalent in psoriasis patients (p = 0.0535, chi-square test, Table 2) [14]. In rs12387, A > G SNPs were significantly more common in our cohort than in “8.3K JPN” (p = 0.0363, chi-square test, Table 3).
Table 2
Genotyping results for rs12529 and comparison with other cohorts.
rs12529
Study
|
Population
|
Sample
number
|
Major Allele
G
|
Minor Allele
C
|
Chi-square test
P value
|
Our study
|
Japanese
|
231
|
400
(86.6%)
|
62
(13.4%)
|
|
ss69068306
|
Japanese
|
90
|
168
(93.3%)
|
12
(6.7%)
|
0.0080
|
ss71643788
|
Japanese
|
88
|
166
(94.3%)
|
10
(5.7%)
|
0.0029
|
8.3KJPN
|
Japanese
|
16760
|
29816
(88.9%)
|
3704
(11.1%)
|
0.0535
|
1000Genomes
|
Global
|
5008
|
5797
(58.0%)
|
4203
(42.0%)
|
|
Table 3
Genotyping results for rs12387 and comparison with other cohorts.
rs12387
Study
|
Population
|
Sample
number
|
Major Allele
A
|
Minor Allele
G
|
Chi-square test
P value
|
Our study
|
Japanese
|
231
|
400
(86.6%)
|
62
(13.4%)
|
|
ss2827707
|
Japanese
|
172
|
316
(91.9%)
|
28
(8.1%)
|
0.0093
|
ss66361131
|
Japanese
|
90
|
168
(94.4%)
|
10
(5.6%)
|
0.0026
|
8.3KJPN
|
Japanese
|
16760
|
29898
(89.2%)
|
3622
(10.8%)
|
0.0363
|
1000Genomes
|
Global
|
5008
|
8482
(84.8%)
|
1518
(15.2%)
|
|
The group with the rs12529 G > C SNP, which also had the rs12387 A > G SNP, had more PV and less PsA (p = 0.0386, Fisher’s exact test, Fig. 1A). No correlation was detected between the AKR1C3 variant and smoking (data not shown), PASI score (Fig. 1B), onset age (Fig. 1C), BMI (Fig. 1D), white blood cell (WBC) count at first visit (Fig. 1E), or number of times biologics were switched (Fig. 1F).
Akr1c3 Snps Are Related To Early-onset Psoriasis In Female Patients
The mean onset age in the cohort was 33.3 years (Table 1), and did not differ significantly between males (33.7 years) and females (32.7 years); the distribution, however, was very different (Fig. 2A). As in previous reports [15, 16], the onset age in female patients had bimodal peaks, with the first peak around 20 years of age and the second peak between 40 and 50 years of age. Female patients with rs12529 G > C and rs12387 A > G SNPs were more common in the first peak (> 22 years of age). The number of female patients with an onset age ≤ 22 having the rs12529 G/C, C/C and rs12387A/G, G/G variants was significantly higher than that of female patients having the rs12529 G/G and rs12387A/A SNPs (p = 0.0213, Fisher’s exact test, Fig. 2B). Young female patients with an onset age of ≤ 22 having rs12529 G > C and rs12387 A > G SNPs had significantly higher PASI scores than those with rs12529 G/G and rs12387 A/A SNPs (p = 0.0063, Student’s test, Fig. 2C). No correlation was detected between clinical features, including WBC count (Fig. 2D), number of times that biologics were switched (Fig. 2E), BMI (Fig. 2F), and AKR1C3 variants, in young female patients or in the whole cohort.
Akr1c3 Expression In The Psoriasis Lesional Epidermis Is Decreased In Patients With Akr1c3 Snps
Immunostaining of AKR1C3 and loricrin was performed on formalin-fixed paraffin-embedded tissues of psoriasis lesions obtained from 62 of 231 patients (45 males, 17 females; 28 patients with PV, 19 with PsA, 11 with GPP, 1 with EP, 3 with complications). AKR1C3 expression (red) in the epidermis from psoriasis lesional skin samples was significantly lower in patients with rs12529 G > C and rs12387 A > G SNPs than in patients with rs12529 G/G and rs12387 A/A SNPs (p = 0.0434, Student’s t-test, Fig. 3A, D). As for loricrin expression (green) in the epidermis, abnormally early and weak, but broad expression was observed in the epidermis from patients with rs12529 G > C and rs12387 A > G SNPs (Fig. 3B, E). Expression of AHR, which induces AKR1C3 [11], also did not differ significantly between patients with different SNPs (Fig. 3C). Representative immunostaining images of AKR1C3 and loricrin staining are shown in Fig. 3D. Ectopic expression of loricrin in the spinous layer in patients with the rs12529 G > C SNP is shown in Fig. 3E. This abnormal early expression of loricrin may be due to the weaker suppression of keratinocyte differentiation by AKR1C3 in patients with the rs12529 G > C SNP.
Akr1c3 Is Downregulated And Loricrin Is Upregulated In Cells Isolated From Patients With Akr1c3 Snps
We isolated epidermal keratinocytes from patients with AKR1C3 rs12529 G/G, G/C, C/C and rs12387 A/A, A/G, G/G, and analyzed them with 2D skin culture. After 6 days of treatment with 2.5 µM benzo[a]pyrene (BaP), a typical air pollutant and AHR agonist, and 200 µM indole-3-carbinol (I3C, an AHR agonist that specifically induces AKR1C3), AKR1C3 expression was significantly higher in keratinocytes with rs12529 G/G than in keratinocytes with rs12529 C/C (with BaP, p = 0.0009, Dunnett’s test, Fig. 4A, with I3C, p = 0.0353 Dunnett’s test, Fig. 4B) by real-time polymerase chain reaction (PCR). Expression of genes related to keratinocyte differentiation after 6-day treatment with 200 µM I3C was analyzed, and expression of genes associated with late differentiation, including involucrin and loricrin, was significantly upregulated in keratinocytes obtained from patients with rs12529 G > C and rs12387 A > G SNPs, despite using 2D cultures with no air-liquid interface (p = 0.0495, Wilcoxon test, Fig. 4C). Interestingly, rs12529 G/C-derived keratinocytes exhibited high loricrin expression, while rs12529 C/C-derived keratinocytes exhibited reduced loricrin expression and the highest involucrin expression.
Abnormal early expression of loricrin observed in 3D skin culture of keratinocytes from patients with AKR1C3 SNPs
The 3D skin culture was constructed with keratinocytes isolated from patients with each of the AKR1C3 SNPs. The incubation period was 2 weeks; for the first 7 days, keratinocytes were incubated in the medium supplied with the EPI-KIT (J-TEC, Gamagori, Japan), and for the second 7 days, they were incubated in medium supplemented with 300 µM I3C. Keratinocytes were fixed on day 14 and immunohistochemical staining was performed. Abnormally early expression of loricrin (green staining) from the basal layer was observed in the skin cultures from patients with rs12529 G/C and C/C SNPs (Fig. 4D).
Expression Landscape Of Genes Associated With Keratinocyte Differentiation In Psoriasis
Analysis was performed using the profiled data set accessible at the GEO database (GSE13355) [17]. Clustering analysis revealed genes that were downregulated and upregulated in psoriasis lesions (Fig. 5A). Loricrin (LOR) was the most significantly downregulated gene in the psoriasis lesions, and filaggrin (FLG, FLG2) was also strongly downregulated. Involucrin (IVL), on the other hand, was upregulated. Keratin 5 (KRT5) was highly expressed while keratin 10 (KRT10) was suppressed. Although tight junction protein 1 (TJP1) and occludin (OCLN) were strongly expressed in lesional skin, the absence of claudin (CLDN1) expression indicates that tight junctions were not well formed. In the psoriasis lesional skin, high expression of desmocollin 2 (DSC2) and desmoglein 3 (DSG3) was also observed, in contrast to the low expression of desmocollin 1 (DSC1) and desmoglein 1 (DSG1). The psoriasis lesional skin was characterized by increased gene expression in the lower to middle epidermis and decreased gene expression related to terminal differentiation in the upper layer of the epidermis and granular layer, and cornification. The expression of AKR1C3 was also included in this data set. In the psoriasis lesional skin, the expression of AKR1C3 was significantly downregulated compared with that in healthy normal skin and non-lesional skin (Fig. 5B). Although the number of AKR1C3 SNPs in this cohort is unknown, keratinocyte differentiation was clearly abnormal in the psoriasis lesional skin. This database differs from our Japanese cohort in that it comprises a US cohort. As described above, however, the rs12529 G > C SNP is much more common in the US, even in healthy individuals.